System for real time detection of nucleic acid amplification products

ABSTRACT

A system is provided for carrying out real time fluorescence-based measurements of nucleic acid amplification products. In a preferred embodiment of the invention, an excitation beam is focused into a reaction mixture through a surface, the reaction mixture containing (i) a first fluorescent indicator capable of generating a first fluorescent signal whose intensity is proportional to the amount of an amplification product in the volume of the reaction mixture illuminated by the excitation beam and (ii) a second fluorescent indicator homogeneously distributed throughout the reaction mixture capable of generating a second fluorescent signal proportional to the volume of reaction mixture illuminated by the excitation beam. Preferably, the excitation beam is focused into the reaction mixture by a lens through a portion of a wall of a closed reaction chamber containing the reaction mixture. The same lens is used to collect the first and second fluorescent signals generated by the first and second fluorescent indicators, respectively, in response to the excitation beam. The ratio of the fluorescent intensities of the first and second fluorescent signals provides a stable quantitative indicator of the amount of amplification product synthesized in the course of the amplification reaction.

This application is a continuation of application Ser. No. 8/235,411,filed Apr. 29, 1994, abandoned.

The invention relates generally to the field of nucleic acidamplification, and more particularly to a system for measuring in realtime polynucleotide products from nucleic acid amplification processes,such as polymerase chain reaction (PCR).

BACKGROUND

Nucleic acid sequence analysis is becoming increasingly important inmany research, medical, and industrial fields, e.g. Caskey, Science 236:1223-1228 (1987); Landegren et al, Science, 242: 229-237 (1988); andArnheim et al, Ann. Rev. Biochem., 61: 131-156 (1992). The developmentof several nucleic acid amplification schemes has played a critical rolein this trend, e.g. polymerase chain reaction (PCR), Innis et al,editors, PCR Protocols (Academic Press, New York, 1990); McPherson etal, editors, PCR: A Practical Approach (IRL Press, Oxford, 1991);ligation-based amplification techniques, Barany, PCR Methods andApplications 1: 5-16 (1991); and the like.

PCR in particular has become a research tool of major importance withapplications in cloning, analysis of genetic expression, DNA sequencing,genetic mapping, drug discovery, and the like, e.g. Arnheim et al (citedabove); Gilliland et al, Proc. Natl. Acad. Sci., 87: 2725-2729 (1990);Bevan et al, PCR Methods and Applications, 1: 222-228 (1992); Green etal, PCR Methods and Applications, 1: 77-90 (1991); Blackwell et al,Science, 250: 1104-1110 (1990).

A wide variety of instrumentation has been developed for carrying outnucleic acid amplifications, particularly PCR, e.g. Johnson et al, U.S.Pat. No. 5,038,852 (computer-controlled thermal cycler); Wittwer et al,Nucleic Acids Research, 17: 4353-4357 (1989)(capillary tube PCR);Hallsby, U.S. Pat. No. 5,187,084 (air-based temperature control); Garneret al, Biotechniques, 14: 112-115 (1993)(high-throughput PCR in 864-wellplates); Wilding et al, International application No. PCT/US93/04039(PCR in micro-machined structures); Schnipelsky et al, European patentapplication No. 90301061.9 (publ. No. 0381501 A2)(disposable, single usePCR device), and the like. Important design goals fundamental to PCRinstrument development have included fine temperature control,minimization of sample-to-sample variability in multi-sample thermalcycling, automation of pre- and post-PCR processing steps, high speedcycling, minimization of sample volumes, real time measurement ofamplification products, minimization of cross-contamination, or samplecarryover, and the like. In particular, the design of instruments thatpermit PCR to be carried out in closed reaction chambers and monitoredin real time is highly desirable. Closed reaction chambers are desirablefor preventing cross-contamination, e.g. Higuchi et al, Biotechnology,10: 413-417 (1992) and 11: 1026-1030 (1993); and Holland et al, Proc.Natl. Acad. Sci., 88: 7276-7280 (1991). Clearly, the successfulrealization of such a design goal would be especially desirable in theanalysis of diagnostic samples, where a high frequency of falsepositives and false negatives would severely reduce the value of thePCR-based procedure. Real time monitoring of a PCR permits far moreaccurate quantitation of starting target DNA concentrations inmultiple-target amplifications, as the relative values of closeconcentrations can be resolved by taking into account the history of therelative concentration values during the PCR. Real time monitoring alsopermits the efficiency of the PCR to be evaluated, which can indicatewhether PCR inhibitors are present in a sample.

Holland et al (cited above) and others have proposed fluorescence-basedapproaches to provide real time measurements of amplification productsduring a PCR. Such approaches have either employed intercalating dyes(such as ethidium bromide) to indicate the amount of double stranded DNApresent, or they have employed probes containing fluorescer-quencherpairs (the so-called "Tac-Man" approach) that are cleaved duringamplification to release a fluorescent product whose concentration isproportional to the amount of double stranded DNA present.

Unfortunately, successful implementation of these approaches has beenimpeded because the required fluorescent measurements must be madeagainst a very high fluorescent background. Thus, even minor sources ofinstrumental noise, such as the formation of condensation in the chamberduring heating and cooling cycles, formation of bubbles in an opticalpath, particles or debris in solution, differences in samplevolumes--and hence, differences in signal emission and absorbence, andthe like, have hampered the reliable measurement of the fluorescentsignals.

In view of the above, it would be advantageous if an apparatus wereavailable which permitted stable and reliable real time measurement offluorescent indicators of amplification products resulting from any ofthe available nucleic acid amplification schemes.

SUMMARY OF THE INVENTION

The invention relates to a system for carrying out real timefluorescence-based measurements of nucleic acid amplification products.In a preferred embodiment of the invention, an excitation beam isfocused into a reaction mixture containing (i) a first fluorescentindicator capable of generating a first fluorescent signal whoseintensity is proportional to the amount of an amplification product inthe volume of the reaction mixture illuminated by the excitation beamand (ii) a second fluorescent indicator homogeneously distributedthroughout the reaction mixture and capable of generating a secondfluorescent signal proportional to the volume of reaction mixtureilluminated by the excitation beam. It is understood that theproportionality of the fluorescent intensities is for a constant set ofparameters such as temperature, pH, salt concentration, and the like,that independently influence the fluorescent emissions of organic dyes.

Preferably, the excitation beam is focused into the reaction mixture bya lens through a portion of a wall of a closed reaction chambercontaining the reaction mixture. In further preference, the same lenscollects the first and second fluorescent signals generated by the firstand second fluorescent indicators, respectively, in response to theexcitation beam; thus, variability in the collected signal due tomisalignment of excitation and collection optics is avoided. In thisembodiment, whenever the lens directs the excitation beam through aportion of a wall of the closed reaction chamber which is not in contactwith the reaction mixture, that portion of the wall is heated so thatcondensation from the reaction mixture does not form in the opticalpathway of the fluorescent signals being collected by the lens, therebyremoving another source of variability in the collected signal.

In the most preferred embodiment, the reaction chamber is a tube with aclosed end, referred to herein as the bottom of the tube, and an openend, referred to herein as the top of the tube, which can be closed witha cap such that a leak-proof seal is formed. In other words, once areaction mixture is placed in the tube and the cap is attached a closedreaction chamber is formed. In this most preferred embodiment, (1) thereaction mixture fills a portion of the tube, generally at the bottom ofthe tube, such that a void is left between the cap of the tube and a topsurface of the reaction mixture, and (2) the lens without contacting thecap focuses the excitation beam through the cap into the reactionmixture through its top surface and collects the resulting fluorescencegenerated by the first and second fluorescent indicators. As mentionedabove, the portion of the tube through which the excitation beampasses--the cap in this embodiment--is heated to prevent the formationof condensation which would introduce an added source of variability inthe measurement of the collected fluorescent signals. Potentialvariability that could arise from sequential analysis of the first andsecond fluorescent signals is eliminated by simultaneously analyzing thesignals by spectrally separating the signal light onto an array of photodetectors, e.g. by diffracting the signal onto a charged-coupled device(CCD) array.

As discussed more fully below, an excitation beam generated by a singlelight source, e.g. a laser, is conveniently distributed to a pluralityof closed reaction chambers by fiber optics. Likewise, the same fiberoptics can collect the fluorescent signals from the plurality ofreaction chambers for analysis by a single detection and analysissystem.

Preferably, the system is employed with the PCR amplification of nucleicacids.

The system of the invention permits accurate real time monitoring ofnucleic amplification reactions by providing apparatus and fluorescentreagents for generating a stable fluorescent signal proportional to theamount of amplification product and independent of variations in thevolume of reaction mixture. The availability of data showing theprogress of amplification reactions leads to more accurate estimates ofrelative starting concentrations of target nucleic acids, to rapidassessment of the efficiency of the amplification reactions, and opensthe possibility of reduced reagent usage and feedback reaction control.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 diagrammatically illustrates a preferred embodiment of the sampleinterface components of the system of the invention.

FIG. 2 diagrammatically illustrates a preferred embodiment forsimultaneously monitoring a plurality of amplification reactions bysequentially interrogating reactions via a fiber optic multiplexer.

FIG. 3 shows spectrally separated fluorescent intensity data for atetramethylrhodamine fluorescent indicator, a fluorescein fluorescentindicator, and instrument background registered by a CCD array of thepreferred embodiment described below.

FIG. 4 shows the time dependence of fluorescent signals from afluorescein dye proportional to the amplification product (firstfluorescent indicator) and a tetramethylrhodamine dye employed as asecond fluorescent indicator during a typical PCR.

FIG. 5 shows the cycle dependence of the ratio of the intensities of thefluorescein and tetramethylrhodamine dyes from the same PCR whose timedependent data is shown in FIG. 3.

FIG. 6 shows data relating the amount of amplification product to cyclenumber in separate PCRs having different starting concentrations of thesame target nucleic acid.

DEFINITIONS

As used herein, the term "stable" in reference to a fluorescent signalmeans that the root means square (RMS) deviation in the signal due tonoise is less than or equal to two percent of the average signalmagnitude. More preferably, stable means that the RMS deviation in thesignal due to noise is less than or equal to one percent of the averagesignal magnitude.

DETAILED DESCRIPTION OF THE INVENTION

The invention is a fluorescence-based system for monitoring in real timethe progress of a nucleic acid amplification reaction. The type ofamplification scheme used with the system is not critical, but generallythe system requires either the use of a nucleic acid polymerase withexonuclease activity or a population of double stranded DNA whichincreases during the course of the reaction being monitored. Exemplaryamplification schemes that may be employed with the system of theinvention include PCR, ligase-based amplification schemes, such asligase chain reaction (LCR), Q-beta replicase-based amplificationschemes, strand displacement amplification (SDA) schemes, such asdescribed by Walker et al, Nucleic Acids Research, 20:1691-1696 (1992),and the like. A comprehensive description of nucleic acid amplificationschemes is provided by Keller and Manak, DNA Probes, Second Edition(Stockton Press, New York, 1993). Fundamental to the system is themeasurement of ratios of fluorescent intensities of a first fluorescentindicator and an internal standard, referred to herein as a secondfluorescent indicator. The first and second fluorescent indicators mustbe spectrally resolvable. That is, their respective emission spectramust be sufficiently non-overlapping so that separate emission peaks areobserved in the combined spectrum. Clearly, the system may begeneralized to include a plurality of first fluorescent indicators, e.g.to monitor the simultaneous amplification of several target nucleicacids in a single reaction, so that a plurality of fluorescent intensityratios are monitored. Several spectrally resolvable dyes suitable foruse in such embodiments are disclosed in Fung et al, U.S. Pat. No.4,855,225; Menchen et al, U.S. Pat. No. 5,188,934; Bergot et al,International Application PCT/JS90/05565; and like references.

The system includes a sample interface--that is, optical componentsoperationally associated with a closed reaction chamber--which comprisesa lens for focusing an excitation beam into the reaction mixture and forcollecting the resulting fluorescence and a fiber optic for transmittingboth the excitation beam from a light source to the lens and thefluorescent signals from the lens to a detection and analysis means.Preferably, the reaction mixture is contained in a closed reactionchamber to prevent cross-sample contamination, or so-called "carryover."The lens therefore focuses the excitation beam and collects fluorescencethrough a portion of a wall of the closed reaction chamber. As mentionedabove, the preferred reaction chamber is a tube, e.g. having thegeometry and volume of a conventional Eppendorf tube. The tube is closedafter the reaction mixture is added by attaching a cap to the open endof the tube. In a preferred embodiment of the sample interface for PCR,the lens directs the excitation beam and collects fluorescence throughthe cap of the tube, as illustrated in FIG. 1. In the illustratedconfiguration, a first end fiber optic 2 is held by ferrule 4, housing6, and plate 10 in a co-axial orientation with lens 8. A second end offiber optic 2 (not shown) is operationally associated with a lightsource and detection and analysis means, discussed more fully below. Thedistance between the end face of fiber optic 2 and lens 8 is determinedby several factors, including the numerical aperture of the fiber optic,the geometry of tube 18, the focal length of lens 8, the diameter oflens 8, and the like. Guidance for selecting values for such variablesin any particular embodiment is readily found in standard texts onoptical design, e.g. Optics Guide 5 (Melles Griot, Irvine, Calif.,1990), or like reference. In the illustrated embodiment, lens 8 has adiameter of 8 mm and is composed of material BK7, available from EdmundScientific (Barrington, N.J.). Fiber optic 2 has a numerical aperture of2. Preferably, the design permits maximal transmission of excitationbeam 28 to reaction mixture 22. For example, lens 8, numerical apertureof fiber optic 2, and the distance between the end of fiber optic 2 andlens 8 are selected so that the diameter of lens 8 equals or exceeds thediameter of excitation beam 28 where beam 28 impinges on the lens (asillustrated in FIG. 1). Excitation beam 28 is focused through cap 16,void 24, and top surface 26 of reaction mixture 22 to a regionapproximately 1-3 times the diameter of the fiber optic just below, e.g.1-3 mm, surface 26. This degree of focusing is not a critical feature ofthe embodiment; it is a consequence of adapting the sample interface tothe geometry and dimensions of a sample holder of a commerciallyavailable thermal cycler. In other embodiments, the geometry anddimension may permit a sharper focus into the reaction mixture.

The lens of the invention may have a variety of shapes depending onparticular embodiments. For example, the lens may be a sphere, truncatedsphere, cylinder, truncated cylinder, oblate spheroid, or truncatedoblate spheroid, or the like, and may be composed of any suitablytransparent refractive material, such as disclosed by Hlousek, U.S. Pat.No. 5,037,199; Hoppe et al, U.S. Pat. No. 4,747,87; Moring et al, U.S.Pat. No. 5,239,360; Hirschfield, U.S. Pat. No. 4,577,109; or likereferences.

Fluorescent light generated by excitation beam 28 is collected by lens 8along approximately the same optical pathway as that defined byexcitation beam 28 and focused onto the end of fiber optic 2 fortransmission to optical separation and analysis components of thesystem.

In further preference, the sample interface also includes means forheating the portion of the wall of the reaction chamber used for opticaltransmission in order to reduce variability due to scatter and/orabsorption of the excitation beam and signal from condensation ofreaction mixture components. In the embodiment of FIG. 1, the portion ofthe reaction chamber (tube 18) wall used for optical transmission is cap16. Accordingly, heating element 12 and heat-conductive platen 14 areemployed to heat cap 16. Preferably, heating element 12 comprisesresistance heating elements and temperature sensors that permitprogrammed controlled of the temperature of cap 16. Cap 16 is maintainedat a temperature above the condensation points of the components of thereaction mixture. Generally, cap 16 may be maintained at a temperaturein the range of 94-110° C. Preferably, cap 16 is maintained at atemperature in the range of about 102° C. to about 105° C. since theprincipal solvent in the reaction mixture is usually water. Morepreferably, cap 16 is maintained at 103° C. Preferably, in embodimentsemploying thermal cycling, the cap-heating components described aboveare thermally isolated from heating-conducting component 20 employed tocyclically control the temperature of reaction mixture 22.

Selection of appropriate materials for the components described above iswell within the skill of an ordinary mechanical engineer. Exemplarycriterion for material selection include (i) degree of thermalexpansion, especially for amplification schemes employing thermalcycling, and its affect on the alignment of the optical components, (ii)optical transmission properties in the excitation wavelengths andfluorophore emission wavelengths employed, (iii) chemical inertness ofthe reaction chamber relative to components of the reaction mixture,(iv) degree to which critical reaction components, e.g. polymerases,target nucleic acids, would tend to adsorb onto chamber walls, (v)minimization of fluorescent materials in the optical pathway, and thelike. Typically, tubes containing amplification reaction mixtures aremade of polypropylene or like materials.

The sample interface shown in FIG. 1 may be employed individually or itmay be employed as one of a plurality of identical interfaces in asingle instrument, as shown diagrammatically in FIG. 2. In theillustrated embodiment, individual sample interfaces 31, arrayed inholder 30 (which may, for example, be a heating block associated withthermal cycler 32, such as described in Mossa et al, European patentapplication No. 91311090.4, publ. No. 0488769 A2) are connected by fiberoptics 34 to fiber optic multiplexer 36, which selectively permitstransmission between individual fiber optics and port 35, e.g. underuser control via a programmed microprocessor. In a preferredconfiguration, excitation beam 41, generated by light source 52 andcontroller 54, passes through beam splitter 40 and is focused onto port35 by lens 38, where it is sequentially directed by fiber opticmultiplexer 36 to each of a predetermined set, or subset, of fiberoptics 34. Conversely, a fluorescent signal generated in a reactionchambers is collected by lens 8 and focused onto a fiber optic which, inturn, transmits the signal to a detection and analysis means, possiblyvia a fiber optic multiplexer. Returning to FIG. 2, a fluorescent signalcollected by a sample interface is directed to fiber optic multiplexer36 where it emerges through port 35 and is collected and collimated bylens 38. Lens 38 directs the fluorescent signal to beam splitter 40which, in turn, selectively directs the signal through cut-off filter42, which prevents light from the excitation beam from reaching thesignal detection components. Beam splitter 40 may be a conventionaldichroic mirror, a fully reflective mirror with an aperture to pass theexcitation beam (e.g. as disclosed in U.S. Pat. No. 4,577,109), or likecomponent. After passing through cut-off filter 42, the fluorescentsignal is directed by lens 44 to a spectral analyzer which spectrallyseparates the fluorescent signal and measures the intensities of aplurality of the spectral components of the signal. Typically, aspectral analyzer comprises means for separating the fluorescent signalinto its spectral components, such as a prism, diffraction grating, orthe like, and an array of photo-detectors, such as a diode array, acharge-coupled device (CCD) system, an array of bandpass filters andphotomultiplier tubes, or the like. In the preferred embodiment of FIG.2, the spectral analyzer comprises diffraction grating 46 (e.g., modelCP-140, Jobin-Yvon, NJ) and CCD array 48 (e.g., model S2135 PrincetonInstruments, NJ), which is linked to CCD controller 50.

An exemplary CCD array suitable for analyzing fluorescent signal fromfluorescein and tetramethylrhodamine is partitioned into 21 collectionbins which span the 500 nm to 650 nm region of the spectrum. Each bincollects light over a 8.5 nm window. Clearly, many alternativeconfigurations may also be employed. An exemplary application of a CCDarray for spectral analysis is described in Karger et al, Nucleic AcidsResearch, 19: 4955-4962 (1991).

Analyzing the fluorescent signal based on data collected by a spectralanalyzer is desirable since components of the signal due to one or morefirst fluorescent indicators and a second fluorescent indicator (fromwhich intensity ratios are calculated) can be analyzed simultaneouslyand without the introduction of wavelength-specific system variabilitythat might arise, e.g. by misalignment, in a system based on multiplebeam splitters, filters, and photomultiplier tubes. Also, a spectralanalyzer permits the use of "virtual filters" or the programmedmanipulation of data generated from the array of photo-detectors,wherein a plurality of discrete wavelength ranges are sampled--inanalogy with physical bandpass filters--under programmable control viaan associated microprocessor. This capability permits a high degree offlexibility in the selection of dyes as first and second fluorescentindicators.

Generally, the detection and analysis means may be any detectionapparatus to provides a readout that reflect the ratio of intensities ofthe signals generated by the first and second fluorescent indicators.Such apparatus is well know in the art, as exemplified by U.S. Pat. Nos.4,577,109 and 4,786,886 and references such as The Photonics Design &Applications Handbook, 39th Edition (Laurin Publishing Co., Pittsfield,Mass., 1993).

Preferably, the system of the invention is employed to monitor PCRs,although it may also be employed with a variety of other amplificationschemes, such as LCR. Descriptions of and guidance for conducting PCRsis provided in an extensive literature on the subject, e.g. includingInnis et al (cited above) and McPherson et al (cited above). Briefly, ina PCR two oligonucleotides are used as primers for a series of syntheticreactions that are catalyzed by a DNA polymerase. These oligonucleotidestypically have different sequences and are complementary to sequencesthat (i) lie on opposite strands of the template, or target, DNA and(ii) flank the segment of DNA that is to be amplified. The target DNA isfirst denatured by heating in the presence of a large molar excess ofeach of the two oligonucleotides and the four deoxynucleosidetriphosphates (dNTPs). The reaction mixture is then cooled to atemperature that allows the oligonucleotide primers to anneal to theirtarget sequences, after which the annealed primers are extended with DNApolymerase. The cycle of denaturation, annealing, and extension is thenrepeated many times, typically 25-35 times. Because the products of oneround of amplification serve as target nucleic acids for the next, eachsuccessive cycle essentially doubles the amount of target DNA, oramplification product.

As mentioned above an important aspect of the invention is thefluorescent dyes used as the first and second fluorescent indicators. Byexamining the ratio of the fluorescent intensities of the indicators,the effects of most sources of systematic variability, which would beapparent in the intensities alone, are eliminated. Generally, inaccordance with the invention, the first fluorescent indicator may be acomplex-forming dye or a dye covalently attached to an oligonucleotideprobe which is degraded during polymerization steps to generate asignal. This later embodiment relates to the so-called "Tacman"approach, described by Holland et al, Proc. Natl. Acad. Sci., 88:7276-7280 (1991). As used herein, the term "complex-forming" inreference to a dye means that a dye is capable of forming a stablenon-covalent complex with either double stranded or triple strandednucleic acid structures, usually DNA, and that the dye's fluorescentcharacteristics are substantially different in the complexed state ascompared to a non-complexed, i.e. usually free-solution, state.Preferably, the quantum efficiency of fluorescence of an complex-formingdye is enhanced in the complexed state as compared to the free-solutionstate, thereby resulting in enhanced fluorescent upon complex formation.Exemplary complex-forming dyes include ethidium bromide, propidiumiodide, thiazole orange, acridine orange, daunomycin, mepacrine,4',6'-diaminidino-2-phenylindole (DAPI), oxazole orange,bisbenzimidaxole dyes, such as Hoechst 33258 and Hoechst 33342, andheterodimers of various intercalating dyes, such as ethidium, acridine,thiazolium, and oxazolium dyes (known by their acronyms POPRO, BOPRO,YOPRO, and TOPRO), and like dyes, which are described in the followingreferences: Haugland, pgs. 221-229 in Handbook of Fluorescent Probes andResearch Chemicals, 5th Edition (Molecular Probes, Inc., Eugene, 1992);Glazer et al, Proc. Natl. Acad. Sci., 87: 3851-3855 (1990); Srinivasanet al, Applied and Theoretical Electrophoresis, 3: 235-239 (1993);Kapuscinski et al, Anal. Biochem., 83: 252-257 (1977); Hill, Anal.Biochem., 70: 635-638 (1976); Setaro et al, Anal. Biochem., 71: 313-317(1976); and Latt et al, J. Histochem. Cytochem., 24:24-33 (1976); andRye et al, Nucleic Acids Research, 20: 2803-2812 (1992). Preferably,when complex-forming dyes are employed as first fluorescent indicators,such dyes are selected from the group consisting of thiazole orange,ethidium bromide, and TOPRO.

Dyes employed as second fluorescent indicators include fluorescent dyeswhose fluorescent characteristics are substantially unaffected by thepresence or association with nucleic acids, particularly double strandedDNA. Such dyes may include virtually any fluorescent dye fulfilling thiscriterion which is also spectrally resolvable from whatever firstfluorescent indicators that are employed. Preferred second fluorescentindicators include rhodamine dyes and fluorescein dyes. More preferably,the second fluorescent indicator is tetramethylrhodamine or2',4',5',7',-tetrachloro-4,7-dichlorofluorescein, the latter beingdisclosed by Menchen et al, U.S. Pat. No. 5,188,934.

In a preferred embodiment, a first fluorescent indicator and a secondfluorescent indicator are both covalently attached to an oligonucleotideprobe as described by Lee et al, Nucleic Acid Research, 21: 3761-3766(1993). More specifically, fluorescein is used as the first fluorescentindicator and tetramethylrhodamine is used as the second fluorescentindicator such that the tetramethylrhodamine moiety substantiallyquenches any fluorescent emissions by the fluorescein moiety. Thus, whenboth dyes are attached to the same oligonucleotide, only thetetramethylrhodamine is capable of generating a fluorescent signal. Whenthe oligonucleotide is cleaved, e.g. via the 5'->3' exonuclease activityof a DNA polymerase, separating the two dyes, the fluorescein becomecapable of generating a fluorescent signal. Preferably, in thisembodiment, the excitation beam is generated from the 488 nm emissionline of an argon ion laser. In accordance with the invention, in a PCRthe production of "free" fluorescein in this embodiment is proportionalto the amount of DNA synthesis catalyzed by the DNA polymerase employed,and hence, the amount of amplification product. In this embodiment,preferably the first fluorescent indicator is fluorescein, e.g. 6-FAM(available from Applied Biosystems, Foster City), and the secondfluorescent indicator is either tetramethylrhodamine or2',4',5',7',-tetrachloro-4,7-dichlorofluorescein.

Such oligonucleotide probes of the invention can be synthesized by anumber of approaches, e.g. Ozaki et al, Nucleic Acids Research, 20:5205-5214 (1992); Agrawal et al, Nucleic Acids Research, 18: 5419-5423(1990); or the like. Preferably, the oligonucleotide probes aresynthesized on an automated solid phase DNA synthesizer usingphosphoramidite chemistry, e.g. Applied Biosystems, Inc. model 392 or394 DNA synthesizer (Foster City, Calif.). The first and secondfluorescent indicators can be covalently attached to predeterminednucleotide of an oligonucleotide by using nucleoside phosphoramiditemonomers containing reactive groups. For example, such reactive groupscan be on a phosphate, or phosphate analog, e.g. Agrawal et al (citedabove), on the 5' hydroxyl when attachment is to the 5' terminalnucleotide, e.g. Fung et al, U.S. Pat. No. 4,757,141 or Hobbs Jr., U.S.Pat. No. 4,997,928, and on base moieties, e.g. as disclosed by Ruth,U.S. Pat. No. 4,948,882; Haralambidis et al, Nucleic Acids Research, 15:4857-4876 (1987); Urdea et al, U.S. Pat. No. 5,093,232; Cruickshank U.S.Pat. No. 5,091,519; Hobbs Jr. et al, U.S. Pat. No. 5,151,507; or thelike. Most preferably, nucleotides having pyrimidine moieties arederivatized. In further preference, the 3' terminal nucleotide of theoligonucleotide probe is blocked or rendered incapable of extension by anucleic acid polymerase. Such blocking is conveniently carried out bythe attachment of a phosphate group, e.g. via reagents described by Hornand Urdea, Tetrahedron Lett., 27: 4705 (1986), and commerciallyavailable as 5' Phosphate-ON™ from Clontech Laboratories (Palo Alto,Calif.). Preferably, the oligonucleotide probe is in the range of 15-60nucleotides in length. More preferably, the oligonucleotide probe is inthe range of 18-30 nucleotides in length.

The separation of the first and second fluorescent indicators within theoligonucleotide probe can vary depending on the nature of the firstfluorescent indicator and second fluorescent indicator, the manner inwhich they are attached, the illumination source, and the like. Guidanceconcerning the selection of an appropriate distance for a givenembodiment is found in numerous references on resonant energy transferbetween fluorescent molecules and quenching molecules (also sometimesreferred to as "donor" molecules and "acceptor" molecules,respectively), e.g. Stryer and Haugland, Proc. Natl. Acad. Sci., 58:719-726 (1967); Clegg, Meth. Enzymol., 211: 353-388 (1992); Cardullo etal, Proc. Natl. Acad. Sci., 85: 8790-8794 (1988); Ozaki et al (citedabove); Haugland (cited above); Heller et al, Fed. Proc., 46: 1968(1987); and the like. The first and second fluorescent indicators mustbe close enough so that substantially all, e.g. 90%, of the fluorescencefrom the first fluorescent indicator is quenched. Typically, for energytransfer-based quenching, the distance between the first and secondfluorescent indicators should be within the range of 10-100 angstroms.Preferably, the first and second fluorescent indicators are separated bybetween about 4 to 10 nucleotides, and more preferably, they areseparated by between 4 and 6 nucleotides, with the proviso that thereare no intervening secondary structures, such as hairpins, or the like.Preferably, either the first or second fluorescent indicator is attachedto the 5' terminal nucleotide of the oligonucleotide probe.

Clearly, related embodiments of the above may be employed wherein thefirst fluorescent indicator is attached to an oligonucleotide probe withanother non-fluorescent quenching molecule, instead of a secondfluorescent indicator. In such embodiments, the second fluorescentindicator could be virtually any spectrally resolvable fluorescent dyethat did not interact with the amplification products.

EXPERIMENTAL Real Time Monitoring of PCR Amplification of DNA Encodingβ-actin from Various Starting Concentrations of Target DNA

A 296 basepair segment of a target DNA encoding human β-actin wasamplified by PCR from various starting amounts in the range of 5×10³ to1×10⁶ copies of target DNA. The following primers and probe wereemployed:

    5'-TCACCCACACTGTGCCCATCTACGA (forward primer)              SEQ ID NO: 1

    5'-CAGCGGAACCGCTCATTGCCAATGGT (reverse primer)             SEQ ID NO: 2

    5'-A(FAM)TGCCCT(TMR)CCCCCATGCCATCCTGCGT (probe)            SEQ ID NO: 3

wherein "FAM" indicates a fluorescein molecule coupled to theoligonucleotide by reacting an NHS-ester group attached to thefluorescein's 6 carbon with a 5'-aminophosphate attached to the5'-terminal deoxyadenosine of the oligonucleotide in accordance withFung et al, U.S. Pat. No. 5,212,304; and wherein "TMR" indicates atetramethylrhodamine molecule coupled to the base moiety of the adjacentthymidine via the amino linking agent disclosed by Urdea et al, U.S.Pat. No. 5,093,232.

PCRs were carried out in 0.2 mL MicroAmp tubes (Perkin-Elmer, Norwalk,Conn.) with the following components: 10 mM Tris-HCl, pH 8.3, 50 mM KCl,3.5 mM MgCl₂, 200 μM each of the nucleoside triphosphates (with dUTPsubstituted for dTTP in accordance with U.S. Pat. No. 5,035,996 toprevent carryover contamination), 300 nM each of forward and reverseprimers, AmpliTaq (Perkin-Elmer, Norwalk, Conn.) at 0.05 U/μL. To thismixture was added 5 μL Raji DNA (Applied Biosystems, Foster City,Calif.) at 10 ng/μL, 5 μL probe at 2 μM, and 1 μL uracil N-glycosylaseat 1 unit/μL to bring the reaction volume to 51 μL. Heating and coolingcycles were carried out in a model 9600 Thermal Cycler (Perkin-Elmer,Norwalk, Conn.) fitted with a sample holder cover containing the sampleinterface components of the invention. The following temperature profilewas employed: hold for 2 minutes at 50° C.; hold for 10 minutes at 95°C.; cycle through the following temperatures 40 times: 92° C. for 15seconds, 54° C. for 15 seconds, 72° C. for 1 minute; then hold at 72° C.

FIG. 3 illustrates data showing the emission spectra of the fluoresceinand tetramethylrhodamine dyes employed as indicators above andfluorescence due to extraneous sources in the system.

FIG. 4 illustrates data showing fluorescein fluorescent intensity andtetramethylrhodamine fluorescent intensity as a function of cyclenumber. The high frequency oscillations in intensity reflect thetemperature dependence of the fluorescent emission of the two dyes. Anincrease in base line fluorescence for both dyes between cycles 10 and28 is a system-based variation. In FIG. 5, which illustrates the ratioof fluorescein-to-tetramethylrhodamine fluorescent intensity from thesame data, the system-based variation is eliminated and the RMS offluctuations in the readout signal, that is, the ratio of fluorescentintensities, is less than 1% of the average magnitude of the measuredratio.

FIG. 6 illustrates data from PCR of the β-actin DNA starting fromamounts ranging from 5000 target molecules to 10⁶ target molecules asindicated in the figure.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES:  3    - (2) INFORMATION FOR SEQ ID NO: 1:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 25 nucleoti - #des              (B) TYPE: nucleic acid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    #1:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #               25 CATC TACGA    - (2) INFORMATION FOR SEQ ID NO: 2:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 26 nucleoti - #des              (B) TYPE: nucleic acid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    #2:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #              26  TGCC AATGGT    - (2) INFORMATION FOR SEQ ID NO: 3:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 26 nucleoti - #des              (B) TYPE: nucleic acid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    #3:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #              26  CATC CTGCGT    __________________________________________________________________________

We claim:
 1. An apparatus for monitoring the formation of a nucleic acidamplification reaction product in real time, the apparatus comprising:asample holder for holding a sample of nucleic acids to be amplified; afiber optic cable for illuminating a volume of the sample with anexcitation beam; a lens co-axially disposed with the fiber optic cablefor focusing the excitation beam into the volume of the sample and forcollecting from the sample and transmitting to the fiber optic cable afirst fluorescent signal whose intensity is proportional to theconcentration of the amplification reaction product and a secondfluorescent signal whose intensity is proportional to the volume of thesample illuminated by the excitation beam; and a detection and analysismechanism for receiving the first and second fluorescent signals fromthe fiber optic cable at a plurality of times during a nucleic acidamplification, the detection and analysis mechanism measuring theintensities of the first and second fluorescent signals at the pluralityof times and producing a plurality of corrected intensity signals, eachcorrected intensity signal corresponding to a relationship between theintensities of the first and second fluorescent signals at a given time.2. The apparatus according to claim 1 wherein the detection and analysismechanism provides a readout corresponding to the plurality of correctedintensity signals as a function of time.
 3. The apparatus according toclaim 1 wherein the apparatus includesa plurality of sample holders forholding a plurality of samples, a plurality of fiber optic cables forilluminating volumes of the plurality of samples, a plurality of lenses,each co-axially disposed with a first end of a fiber optic cable forfocusing an excitation beam into a sample, and a fiber optic multiplexerwhich couples the detection and analysis mechanism to a second end ofeach of the plurality of fiber optic cables.
 4. The apparatus accordingto claim 1 wherein the sample holder includes a removable reactionchamber for holding the sample.
 5. The apparatus according to claim 4wherein the removable reaction chamber is sealable.
 6. The apparatusaccording to claim 1 wherein the sample holder includes a sealablereaction chamber for holding the sample.
 7. The apparatus according toclaim 1 wherein the sample holder includes an optical interface throughwhich the excitation beam is transmitted from the lens into the sample.8. The apparatus according to claim 7 wherein the sample holder includesa sealable reaction chamber for holding the sample, the opticalinterface forming a wall of the reaction chamber.
 9. The apparatusaccording to claim 7 wherein the apparatus further includes a mechanismfor heating the optical interface to prevent condensation of the sampleon the optical interface.
 10. The apparatus according to claim 9 whereinthe sample holder includes a sealable reaction chamber for holding thesample, the optical interface forming a wall of the reaction chamber.11. The apparatus according to claim 7 wherein the sample holderincludes a removable reaction chamber for holding the sample, theoptical interface forming a wall of the reaction chamber which covers atleast a portion of the sample and which is separated from the sample byan air gap.
 12. A method for monitoring the formation of a nucleic acidamplification reaction product in real time comprising:taking a sampleholder containing a nucleic acid sequence to be amplified to form anucleic acid amplification reaction product, a first fluorescentindicator which produces a first fluorescent signal when illuminated byan excitation beam whose intensity is proportional to a concentration ofthe amplification reaction product in the sample, and a secondfluorescent indicator which produces a second fluorescent signal whenilluminated by the excitation beam whose intensity is proportional to avolume of sample illuminated by the excitation beam; performing anamplification of the nucleic acid sequence in the sample holder;transmitting an excitation beam into the sample holder at a plurality oftimes during the amplification and measuring the intensities of thefirst and second fluorescent signals at the plurality of times; andmonitoring the formation of the nucleic acid amplification reactionproduct in real time by calculating a plurality of corrected intensitysignals, each corrected intensity signal corresponding to a relationshipbetween the intensity of the first and second fluorescent signalsmeasured at the plurality of times, a change in the corrected intensitysignal over time indicating the formation of the nucleic acidamplification reaction product.
 13. The method according to claim 12wherein the first fluorescent indicator is a complex-forming dye. 14.The method according to claim 12, further including the step of sealingthe sample within the sample holder prior to transmitting an excitationbeam into the sample holder.
 15. The method according to claim 12wherein the sample holder includes an optical interface through whichthe excitation beam is transmitted to the sample, the sample holder alsoincluding an air gap separating the optical interface from the sample,the method further including the step of heating the optical interfaceto prevent condensation of the sample on the optical interface.
 16. Themethod according to claim 15, further including the step of sealing thesample within the sample holder prior to transmitting an excitation beaminto the sample.
 17. The method according to claim 12 wherein the stepof taking a sample holder includesadding a sample to a reaction chamberwhich is removable from the sample holder; and adding the removablereaction chamber to the sample holder.
 18. The method according to claim17, further including the step of sealing the sample within theremovable reaction chamber.
 19. The method according to claim 17 whereinthe removable reaction chamber includes an optical interface throughwhich the excitation beam is transmitted from the lens to the sample andan air gap separating the optical interface from the sample, the methodfurther including the step of heating the optical interface to preventcondensation of the sample on the optical interface.
 20. The methodaccording to claim 12 wherein performing the amplification includesperforming at least one cycle of a polymerase chain reaction.
 21. Themethod according to claim 12 wherein performing the amplificationincludes performing at least one cycle of a ligase chain reaction.
 22. Amethod for monitoring the formation of multiple nucleic acidamplification reaction products in real time comprising:taking multiplesample holders, each sample holder containing a nucleic acid sequence tobe amplified to form a nucleic acid amplification reaction product, afirst fluorescent indicator which produces a first fluorescent signalwhen illuminated by an excitation beam whose intensity is proportionalto a concentration of the amplification reaction product in the sample,and a second fluorescent indicator which produces a second fluorescentsignal when illuminated by the excitation beam whose intensity isproportional to a volume of sample illuminated by the excitation beam;performing an amplification of the nucleic acid sequences in themultiple sample holders; transmitting an excitation beam into themultiple sample holders at a plurality of times during the amplificationand measuring the intensities of the first and second fluorescentsignals at the plurality of times; and monitoring the formation ofnucleic acid amplification reaction products in the multiple sampleholders in real time by calculating a plurality of corrected intensitysignals, each corrected intensity signal corresponding to a relationshipbetween the intensity of the first and second fluorescent signalsmeasured at the plurality of times, a change in the corrected intensitysignal over time indicating the formation of the nucleic acidamplification reaction product.
 23. A method for monitoring theformation of multiple nucleic acid amplification reaction products inreal time comprising:taking a sample holder containing a plurality ofnucleic acid sequences to be amplified to form a plurality of nucleicacid amplification reaction products, a plurality of first fluorescentindicators which produce a first fluorescent signal when illuminated byan excitation beam whose intensity is proportional to a concentration ofthe amplification reaction product in the sample, and a secondfluorescent indicator which produces a second fluorescent signal whenilluminated by the excitation beam whose intensity is proportional to avolume of sample illuminated by the excitation beam; performing anamplification of the nucleic acid sequence in the sample holder;transmitting an excitation beam into the sample holder at a plurality oftimes during the amplification and measuring the intensities of thefirst and second fluorescent signals at the plurality of times; andmonitoring the formation of the plurality of nucleic acid amplificationreaction products in the sample holder in real time by calculating aplurality of corrected intensity signals for each of the plurality ofnucleic acid sequences in the sample holder at the plurality of times,each corrected intensity signal corresponding to a relationship betweenthe intensity of the first and second fluorescent signals measured atthe plurality of times, a change in the corrected intensity signal overtime indicating the formation of the nucleic acid amplification reactionproduct.
 24. A method for monitoring the formation of a nucleic acidamplification reaction product in real time comprising:taking a sampleholder containing a nucleic acid sequence to be amplified to form anucleic acid amplification reaction product, and first and secondfluorescent indicators covalently attached to an oligonucleotide capableof hybridizing to the amplification reaction product, the firstfluorescent indicator producing a first fluorescent signal whenilluminated by the excitation beam whose intensity is proportional tothe concentration of amplification reaction product in the sample, thesecond fluorescent indicator producing a second fluorescent signal whenilluminated by the excitation beam whose intensity is proportional tothe volume of the sample illuminated by the excitation beam, the secondfluorescent indicator also quenching the fluorescence of the firstfluorescent indicator; performing an amplification of the nucleic acidsequence in the sample holder; transmitting an excitation beam into thesample holder at a plurality of times during the amplification andmeasuring the intensities of the first and second fluorescent signals atthe plurality of times; and monitoring the formation of the nucleic acidamplification reaction product in real time by calculating a pluralityof corrected intensity signals, each corrected intensity signalcorresponding to a relationship between the intensity of the first andsecond fluorescent signals at the plurality of times, a change in thecorrected intensity signal over time indicating the formation of thenucleic acid amplification reaction product.
 25. The apparatus accordingto claim 1 whereinthe nucleic acid amplification includes a plurality ofamplification cycles; and the detection and analysis mechanism receivesthe first and second fluorescent signals from the fiber optic cable atleast once per amplification cycle and measures the intensities of thefirst and second fluorescent signals at least once per amplificationcycle.
 26. The apparatus according to claim 25 wherein the detection andanalysis mechanism produces at least one corrected intensity signal peramplification cycle.
 27. The method according to claim 12whereinperforming the amplification includes performing a plurality ofcycles of an amplification reaction; and transmitting an excitation beaminto the sample holder at a plurality of times during the amplificationincludes transmitting the excitation beam at least once peramplification cycle.
 28. The method according to claim 27whereinmonitoring the formation of the nucleic acid amplificationreaction product includes calculating at least one corrected intensitysignal per amplification cycle.